The assay of glucose in biological fluid, such as blood or urine is one of the important items in the diagnosis of diabetes, hypoglycemia, or other diseases, or the observation of progress in treatment. Nowadays, the methods for assaying glucose in clinical examinations may be roughly classified into reduction methods, enzymatic methods, condensation methods, simplified assay methods, and so on. The reduction methods have disadvantages in that other reduced substances in addition to glucose are measured. Thus, the enzymatic methods (glucose oxidase method), which are superior in specificity, were introduced in the 1950s. Further, the HK-G6PDH method using hexokinase (hereinafter referred to as HK) and glucose-6-phosphate dehydrogenase (hereinafter referred to as G6PDH) has been accepted as a standard method for assaying glucose with more specific manner.
Although the HK-G6PDH method is useful for obtaining accurate glucose level, the components of the reagent are unstable substances, and thus, a long term storage is difficult. Therefore, the enzymes, substrates and the like are generally supplied in the form of lyophilized products and used after dissolved in a buffer or the like at the time of use. However, there is a demand for stability of the reagent after dissolved, in view of the requirements for workability and cost. For example, Japanese Unexamined Patent Publication (Kokai) No. 56-140899 discloses a technique relating to the stability of a reagent for assaying glucose.
The above-mentioned Patent Publication (Kokai) No. 56-140899 describes a technique for enhancing the stability of the reagent by incorporating a sulfhydryl compound (for example, a reduced type of glutathione and N-acetyl cysteine) and/or a chelating agent (for example, EDTA, EGTA) and a bactericide and a pH buffer. The technique basically focuses on a reagent prepared by dissolving lyophilized products and improves the stability of the aqueous solution (solution B) of the components other than the coenzymes. The stability is maintained for at most about 10 days.
Further, Japanese Unexamined Patent Publication (Kokai) No. 63-24900 discloses a technique relating to a stable liquid enzyme composition for assaying glucose which can be stored for a long period of time (over one year). The reagent is a two-component reagent composed of an enzymatic component (hexokinase and G6PDH) and a coenzymatic component (ATP and NAD). The enzymatic component comprises approximately 20 to 40% (v/v) of a polyol organic solvent, a stabilizer such as EDTA, antioxidant such as albumin or poly(vinylpyrrolidone), and an agent for controlling a microorganism, such as sodium azide, and the pH is adjusted to approximately 7.5. Further, the coenzymatic component contains approximately 5 to 20% (v/v) of a polyol organic solvent, and the pH is adjusted to about 7.5.
However, the reagent per se containing a polyol (such as glycerol) is viscous. Further, if poly (vinylpyrrolidone) is added as an antioxidant, the viscosity of the reagent becomes higher. The high viscosity causes troubles in automatic analyzers, for example, a reduction of the accuracy of tile amount of the reagents added. Further, there is the problem that the stability of the coenzymes, i.e., NAD and ATP, is insufficient.
Recently, it is desired to improve the workability for users, by providing the reagents in the form of liquid from a supplier. Further, such reagents are in many cases used in automatic analyzers, and therefore, it is necessary to reformulate the reagents as a two-component form and maintain the stability of the reagent compositions for a long period of time (for example, from half a year to one year).
The reaction formulas of the above HK-G6PDH method are as follows: ##STR1##
In the above formulas, ATP stands for adenosine 5'-triphosphate, ADP stands for adenosine 5'-diphosphate, and NADP.sup.+ stands for the oxidized form of nicotinamide adenine dinucleotide phosphate.
Each of the components participating the above reactions has stability conditions different from each other. Therefore, depending on the combination of the components, the stability of the reagent for assaying glucose prepared by mixing the components sometimes becomes synergistically improved or sometimes conversely becomes worse.
Further, although the assay hitherto was manually performed by mixing a sample with a one-component reagent, a two-component reagent has recently become usual due to the spread of automatic analyzers. In most cases of such two-component reagents, an initiating reagent (second reagent) contains ATP which is the substrate of the initiating.sub.-- enzyme, and a first reagent contains all the remaining components. In some cases, an enzyme, such as HK or G6PDH (or both enzymes) is used as the second reagent, but there was a problem in the stability of the enzymes. Further, a sufficient storability was not able to be obtained even when ATP was used as the second reagent.
The stability of the enzymes has been improved by removing the interfering contaminant enzymes by sophisticated purification of the enzymes per se and by using heat resistant enzymes, for example, glucokinase (hereinafter referred to as GlcK) derived from Batillus stearothermophilus, HK obtained from recombinant yeast or chemically modified enzymes thereof, but it was difficult to obtain a good storage stability for liquid reagents as a whole.
Of the components other than the stabilized enzymes, each of coenzyme NADP.sup.+ and the substrate ATP are one of the greatest destabilizing factors. In particular, the deterioration of NADP.sup.+ is a factor obstructing storage stability.
The inventors of the present invention engaged in various in-depth studies to solve the above problems in the prior art and as a result discovered a reagent composition having sufficient storage stability as a liquid reagent. The present invention is based on this discovery.